This invention provides derivatives of biologically useful cyclic and acyclic peptides in which one or more amino acid side chains or a segment attached to the peptide chain contain chelating moieties that can tightly bind metal ions, including radionuclides. The labeled peptides carry the metal to specific in vivo targets such as receptors and antigens, and are useful for radiodiagnostic imaging, therapy and radiotherapy. New methods for preparing the peptides are also provided.
Radiolabeled peptides are useful in the diagnosis and therapy of a variety of human disease states that are characterized by overexpression of peptide hormone receptors. Thus, for example, it has been shown that radiolabeled analogues of LHRH (luteinizing hormone releasing hormone) and somatostatin selectively bind to hormone-sensitive tumors characterized by cell-surface overexpression of LHRH hormone receptors. Similarly, peptide hormone analogues such as .sup.123 I-vasoactive intestinal peptide (VIP) , .sup.99m Tc-P829, .sup.111 In-DTPA Octreotide and .sup.111 In-bisMSH-DTPA have been used to image human tumors that over express VIP, somatostatin, somatostatin and melanocyte stimulating hormone (MSH) receptors respectively. See: Virgolini et al. Engl. J. Med. 169:1116 (1994); Virgolini et al. J. Nucl. Med. 36:1732, (1995); Lister-James et al. Nucl. Med., 36, 91P, #370, 1995 meeting abstract; Pearson et al. J. Med. Chem. 39:1361, (1996); Krenning et al. J. Nucl. Med., 33:652 (1992); and Wraight et al. Brit. J. Radiol. 65:112 (1992).
Many tyrosine-containing peptides may be labeled with .sup.125 I by well known methods and used for receptor binding studies. For example, the incidence of VIP receptor upregulation has been studied in vitro in a wide range of cancer types using .sup.125 I-[Tyr.sup.10 ]-VIP as the radioligand. See Reubi, Nucl. Med. 36:1846 (1995). The VIP receptor was detected in a wide variety of cancer types, including breast, prostate, ovarian, pancreatic, endometrial, bladder, colon, esophageal, SCLC, astrocytoma, glioblastoma, meningioma, pheochromocytoma, lymphoma, neuroblastoma adenoma, and GEP tumors. An iodinated VIP analogue .sup.123 I-[Tyr.sup.10 ]-VIP has also been used to image VIP receptor-rich tumors in humans. See Virgolini et al, supra.
The use of radioiodine for in vivo diagnostic and therapeutic uses has distinct disadvantages, however. .sup.123 I, the most useful isotope in vivo, is very expensive ($45.30/mCi) and must be produced in a cyclotron. This isotope, furthermore, has a half-life of only 13.2 hours, thereby requiring that it be produced in a geographic location close to where any radioiodinated imaging agent must be used. Other radioisotopes, such as .sup.99m Tc and .sup.188 Re are preferred for diagnostic and therapeutic uses, respectively. .sup.99m Tc, for example, is inexpensive ($0.50/mCi), is readily available (produced in a generator from .sup.99 Mo, a reactor product) , and has an ideal gamma emission energy for imaging with a gamma camera.
Some peptides either directly contain, or are amenable to the introduction of, residues that allow direct binding of radiometals such as .sup.99m Tc and .sup.188 Re to the peptide. For example, somatostatin contains a disulfide bond that, upon reduction, provides two sulfhydryl-containing cysteine side chains that can directly bind .sup.99m Tc. See U.S. Pat. No. 5,225,180. See also WO 94/28942, WO 93/21962 and WO 94/23758. Complexes of this type tend, however, to be heterogeneous and unstable, which limits their clinical utility. Moreover, the use of free sulfhydryls in this manner limits the radiometals which can be used to label the peptide to those that tightly bind free S-H groups. This method further suffers from the problem that direct binding of the metal to an amino acid side chain can greatly influence the peptide conformation, thereby deleteriously altering the receptor binding properties of the compound.
Most peptides either do not contain a metal-binding amino acid sequence motif or, for various reasons such as those described supra, are not amenable to suitable sequence modifications that would permit introduction of such a motif. Some means of rendering the peptide capable of binding radiometals must therefore be introduced into the peptide. A preferred approach is to attach a metal binding ligand to a specified site within the peptide so that a single defined, stable, complex is formed. The ligands used to bind metals often contain a variety of heteroatoms such as nitrogen, sulfur, phosphorous, and oxygen that have a high affinity for metals.
Chelates have conventionally been attached via covalent linkages to the N-terminus of a peptide or peptide analogue, following independent synthesis of the peptide and chelate moieties. For example, Maina et al. have described the coupling of a tetra-amine chelator to the N-terminus of a somatostatin analogue, allowing .sup.99m Tc labeling of the peptide. See J. Nucl. Biol. Med. 38:452 (1994). Coupling in this manner is, however, undesirable when the N-terminus of the peptide plays an important role in its receptor binding properties. Accordingly, application of this method is limited by the requirement that the N-terminus of the peptide accommodate the presence of a (usually sterically bulky) chelator without deleteriously affecting the binding properties of the peptide.
Alternatively, chelating agents have been introduced into peptide side chains by means of site-selective reactions involving particular amino acid residues. For example, the lysine residue at position 6 of LHRH has been directly acylated with a chelating group. See Bajusz, S. et al. Proc. Natl. Acad. Sci. USA 86:6313 (1989). This method is inherently limited by the lack of chemical selectivity available when more than one side chain can potentially react with the chelator, or when the peptide sequence does not contain an amino acid that can be derivatized in this way. A further limitation of this approach can arise when multidentate ligands are used. A single ligand molecule can react with multiple peptide molecules resulting in the formation of significant amounts of cross-linked products.
Chelating agents have been introduced on the side chain of a peptide through tris amino acids as described by Dunn T. J. et al. WO 94/26294. This method does not provide a method for cyclizing the peptides. The side chain protecting groups used to introduce the ligand described in this work are the same as those typically used for peptide amide cyclization. See Felix et al. Int. J. Peptide Protein Res. 32:441 (1988).
A fully protected BAT (bisaminothiol) chelating agent has been synthesized and coupled to the side chain of a lysine residue, which could then be incorporated into a peptide. See Dean et al. WO 93/25244. These fully protected precursors are very time consuming, expensive and cumbersome to prepare. The difficulty and expense of preparing such precursors make this method untenable for preparing a diverse array of ligands attached to the variety of linkers that is needed to design a metal carrying targeting agent.
One potential solution to this problem is to use a protecting group strategy that allows selective coupling of a chelator moiety to specified positions within a peptide chain. The diversity of chemical reactivities present within the amino acid side chains of a peptide has, however, led to difficulties in achieving sufficient selectivity in site-specific deprotection of protecting groups. This lack of selectivity has also heretofore hampered efforts to selectively deprotect two or more different functional groups within a peptide to allow coupling of these groups in, for example, a cyclic peptide.
Edwards et al. J. Med. Chem. 37:3749 (1994) have disclosed a fragment method of assembling a cyclic disulfide on a resin with a subsequent attachment of an intact ligand (DTPA). This approach afforded the known somatostatin targeting agent DTPA-Octreotide. This approach was specifically designed for the preparation of a known compound. A more typical situation, however, requires that a variety of labeled peptides to optimize binding to a particular target. Such a situation requires, therefore, a broader approach allowing the assembly of multiple ligands, best assembled in fragments, placed at any point desired in a sequence which can also be cyclized at a variety of positions in the peptide sequence.
Additional considerations for the synthesis of peptides that can selectively bind metals include the effect of the chelate on the conformation of the peptide. Most peptides are highly conformationally flexible, whereas efficient receptor binding usually requires that a peptide adopt a specific conformation. Whether or not the peptide can adopt this specific conformation is greatly influenced by charge and hydrophilic/hydrophobic interactions, including the effects of a covalently attached metal chelating moiety. It is possible to enhance peptide receptor affinity and selectivity by restricting the conformations that the peptide can adopt, preferably locking the peptide into an active conformation. This is often most readily achieved by preparing cyclic peptides. Cyclic peptides have the added advantage of enhanced resistance to proteases, and therefore frequently demonstrate a longer biological half-life than a corresponding linear peptide.
Peptides can be cyclized by a variety of methods such as formation of disulfides, sulfides and, especially, lactam formation between carboxyl and amino functions of the N- and C-termini or amino acid side chains. However, the plethora of functionality within a peptide chain typically means that, for all but the shortest peptides, selective coupling between two desired functional groups within a peptide is very difficult to achieve.
It is apparent, therefore, that cyclic peptides that can chelate metals ions while retaining the ability to specifically bind with high affinity to a receptor are greatly to be desired. It is also desirable to have a means of attaching a chelating moiety to any predetermined position within a peptide, and to have a means of selectively forming cyclic peptides between any two preselected positions within a peptide chain. Additionally, it is desirable to have access to a method that would allow a chelating moiety to be coupled to a peptide at any desired stage during peptide synthesis.